ABSTRACT Many intracellular targets are not susceptible to small molecule drugs because they lack natural ligands or even ligand binding sites. Moreover, even if a small molecule drug can bind a desirable target protein, it may not be effective in inhibiting protein function. Small molecules drugs capable of disrupting interactions between two proteins have been particularly difficult to identify. Further, even in cases where a small molecule drug is identified, it must be capable of reaching its target site with good pharmacokinetic properties and minimal off- target toxicity. These stringent requirements have led to long development times and a very small fraction of small molecule drugs that have been successfully translated into the clinic, despite decades of research and countless high-throughput screens. Therapeutic monoclonal antibodies have had considerable success as cancer therapeutics, but their inability to cross cell membranes has restricted their targets to secreted or membrane-associated antigens. If antibodies could be efficiently delivered into the cytosol of living cells, it would significantly increase the number of possible druggable targets. Antibodies can be developed to bind nearly any exposed protein epitope, with high specificity and affinity. There are a countless number of therapeutic possibilities that could be pursued if antibodies could be effectively delivered into cells, from inhibiting protein function, to driving proteins interactions, to tagging proteins for proteosomal degradation. Not surprisingly, numerous attempts have been made to deliver antibodies into cells, but a robust and efficient approach has yet to be identified. The overall goal of this proposal is to develop a modular approach to efficiently deliver antibodies into the cytoplasm of living cells. Recently, we developed a novel bioconjugation strategy that enables the site-specific and covalent attachment of small molecules, proteins, and enzymes to IgG. Utilizing this technology, we screened a variety of IgG conjugates in their ability to be delivered into the cytosol of living cells and identified conjugates that could be cytoplasmically delivered with a ~60% efficiency at sub-micromolar concentrations of IgG with minimal cytotoxicity. The modular nature of our approach not only allows for any `off-the-shelf' IgG to be easily swapped into our system, but also preserves the binding affinity of the IgG variable region. In this proposal, we plan to further optimize our antibody-delivery technology and evaluate the ability of antibodies delivered into the cytoplasm to inhibit normal intracellular function or target intracellular proteins for degradation. The specific aims for this proposal are: Aim 1. Optimize IgG conjugates and conditions for maximum cytoplasmic delivery; Aim 2. Develop IgG conjugates that can target intracellular proteins for degradation; Aim 3. Develop and optimize formulations for cytoplasmic delivery of antibodies in vivo.